Non-destructive bilayer monitoring using measurement of bilayer response to electrical stimulus

ABSTRACT

A method of detecting a lipid bilayer formed in a cell of a nanopore based sequencing chip is disclosed. An integrating capacitor is coupled with a lipid membrane, wherein the lipid membrane is between a working electrode and a counter electrode. An alternating current (AC) voltage is applied to the counter electrode. A voltage across the integrating capacitor is periodically sampled by an analog-to-digital converter (ADC). A change in the sampled voltage across the integrating capacitor in response to a change in the AC voltage is determined. Whether the lipid membrane comprises a lipid bilayer is detected based on the determined change in the sampled voltage across the integrating capacitor in response to the change in the AC voltage.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip.

FIG. 6A illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the circuitry can be configuredto detect whether a lipid bilayer is formed in the cell without causingan already formed lipid bilayer to break down.

FIG. 6B illustrates the same circuitry 600 in a cell of a nanopore basedsequencing chip as that shown in FIG. 6A. Comparing to FIG. 6A, insteadof showing a lipid membrane/bilayer between the working electrode andthe counter electrode, an electrical model representing the electricalproperties of the working electrode and the lipid membrane/bilayer isshown.

FIG. 7 illustrates an electrical model 700 representing the electricalproperties of a portion of circuitry 600 during the lipid bilayermeasurement phase of the system.

FIG. 8A illustrates that a small observed ±ΔV_(ADC) in response to a±ΔV_(liq) detects that no lipid bilayer has been formed in the cell.

FIG. 8B illustrates that a large observed ±ΔV_(ADC) in response to a±ΔV_(liq) detects that a lipid bilayer has been formed in a cell.

FIG. 9A illustrates an exemplary plot of V_(ADC) versus time before andafter a lipid bilayer is formed within a cell.

FIG. 9B illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t₁ when a lipid bilayerhas not been formed.

FIG. 9C illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t₂ when a lipid bilayerhas been formed.

FIG. 10 illustrates an embodiment of a process 1000 for an improvedtechnique of forming lipid layers in the cells of a nanopore basedsequencing chip.

FIG. 11 illustrates the top view of a nanopore based sequencing system1100 with an improved flow chamber enclosing a silicon chip that allowsliquids and gases to pass over and contact sensors on the chip surface.

FIG. 12A illustrates the initial distribution of cells with differentΔV_(ADC) values.

FIG. 12B illustrates the distribution of cells with different ΔV_(ADC)values after the lipid-thinning stimulus phase and salt buffer solutionflowing phase of process 1000 have repeated a number of times.

FIG. 12C illustrates the distribution of cells with different ΔV_(ADC)values after the lipid-thinning stimulus phase and salt buffer solutionflowing phase of process 1000 have repeated an even greater number oftimes.

FIG. 13 illustrates an embodiment of a timing diagram for a bilayermeasurement phase, an electrical lipid-thinning stimulus phase, and asalt buffer solution flowing phase.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. Ananopore based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing soluble protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly onto the surface of the cell. A single PNTMC 104 is insertedinto membrane 102 by electroporation. The individual membranes in thearray are neither chemically nor electrically connected to each other.Thus, each cell in the array is an independent sequencing machine,producing data unique to the single polymer molecule associated with thePNTMC. PNTMC 104 operates on the analytes and modulates the ioniccurrent through the otherwise impermeable bilayer.

With continued reference to FIG. 1, analog measurement circuitry 112 isconnected to a metal electrode 110 covered by a thin film of electrolyte108. The thin film of electrolyte 108 is isolated from the bulkelectrolyte 114 by the ion-impermeable membrane 102. PNTMC 104 crossesmembrane 102 and provides the only path for ionic current to flow fromthe bulk liquid to working electrode 110. The cell also includes acounter electrode (CE) 116, which is an electrochemical potentialsensor. The cell also includes a reference electrode 117.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. Stage A illustrates the components asdescribed in FIG. 3. Stage C shows the tag loaded into the nanopore. A“loaded” tag may be one that is positioned in and/or remains in or nearthe nanopore for an appreciable amount of time, e.g., 0.1 millisecond(ms) to 10000 ms. In some cases, a tag that is pre-loaded is loaded inthe nanopore prior to being released from the nucleotide. In someinstances, a tag is pre-loaded if the probability of the tag passingthrough (and/or being detected by) the nanopore after being releasedupon a nucleotide incorporation event is suitably high, e.g., 90% to99%.

At stage A, a tagged nucleotide (one of four different types: A, T, G,or C) is not associated with the polymerase. At stage B, a taggednucleotide is associated with the polymerase. At stage C, the polymeraseis docked to the nanopore. The tag is pulled into the nanopore duringdocking by an electrical force, such as a force generated in thepresence of an electric field generated by a voltage applied across themembrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage D. For example, a non-paired nucleotide isrejected by the polymerase at stage B or shortly after the processenters stage C.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 picosiemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS, corresponding toone of the four types of tagged nucleotides respectively. The polymeraseundergoes an isomerization and a transphosphorylation reaction toincorporate the nucleotide into the growing nucleic acid molecule andrelease the tag molecule. In particular, as the tag is held in thenanopore, a unique conductance signal (e.g., see signal 210 in FIG. 2)is generated due to the tag's distinct chemical structures, therebyidentifying the added base electronically. Repeating the cycle (i.e.,stage A through E or stage A through F) allows for the sequencing of thenucleic acid molecule. At stage D, the released tag passes through thenanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip. Cell 500 includes a dielectric layer 501. Dielectricmaterial used to form dielectric layer 501 includes glass, oxides,nitrides, and the like. Cell 500 further includes a dielectric layer 504above dielectric layer 501. Dielectric layer 504 forms the wallssurrounding a well 505 in which a working electrode 502 is located atthe bottom. Dielectric material used to form dielectric layer 504includes glass, oxide, silicon mononitride (SiN), and the like. The topsurface of dielectric layer 504 may be silanized. Silanization forms ahydrophobic layer 520 above the top surface of dielectric layer 504. Insome embodiments, hydrophobic layer 520 has a thickness of about 1.5nanometer (nm).

Well 505 formed by the dielectric layer walls 504 further includes afilm of salt solution 506 above working electrode 502. Salt solution 506may include one of the following: lithium chloride (LiCl), sodiumchloride (NaCl), potassium chloride (KCl), lithium glutamate, sodiumglutamate, potassium glutamate, lithium acetate, sodium acetate,potassium acetate, calcium chloride (CaCl₂), strontium chloride (SrCl₂),Manganese chloride (MnCl₂), and magnesium chloride (MgCl₂). In someembodiments, the film of salt solution 506 has a thickness of aboutthree microns (μm).

As shown in FIG. 5, a membrane is formed on top of dielectric layer 504and spans across well 505. For example, the membrane includes a lipidmonolayer 518 formed on top of hydrophobic layer 520. As the membranereaches the opening of well 505, the lipid monolayer transitions to alipid bilayer 514 that spans across the opening of the well. A bulkelectrolyte 508 containing protein nanopore transmembrane molecularcomplexes (PNTMC) and the analyte of interest is placed directly abovethe well. A single PNTMC/nanopore 516 is inserted into lipid bilayer 514by electroporation. Nanopore 516 crosses lipid bilayer 514 and providesthe only path for ionic flow from bulk electrolyte 508 to workingelectrode 502. Bulk electrolyte 508 may further include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂).

Cell 500 includes a counter electrode (CE) 510, which is anelectrochemical potential sensor. Cell 500 also includes a referenceelectrode 512. In some embodiments, counter electrode 510 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 502 is a metal electrode. Fornon-faradaic conduction, working electrode 502 may be made of metalsthat are resistant to corrosion and oxidation, e.g., platinum, gold,titanium nitride and graphite. For example, working electrode 502 may bea platinum electrode with electroplated platinum. In another example,working electrode 502 may be a titanium nitride (TiN) working electrode.

The step of inserting a nanopore into a lipid bilayer is performed afterit is determined that a lipid bilayer has been properly formed within acell of the nanopore based sequencing chip. In some techniques, theprocess of determining whether a lipid bilayer has been properly formedin a cell may cause an already properly formed lipid bilayer to bedestroyed. For example, a stimulus voltage may be applied to cause acurrent to flow across the electrodes. Although the measured response tothe stimulus voltage may be used to distinguish between a cell with aproperly formed lipid bilayer (i.e., a lipid bilayer that is two layersof lipid molecules thick) from a cell without a properly formed lipidbilayer (e.g., a cell with a thick lipid and solvent combined film thatspans across the well of the cell), the stimulus voltage level is highenough to cause an already properly formed lipid bilayer to break downin some instances. In other words, the stimulus voltage for testing thelipid bilayer may be destructive to the lipid bilayer. In the event thatan already properly formed lipid bilayer is destroyed by the stimulusvoltage, a very high current begins to flow across the electrodes as aresult of the short-circuit condition. In response, the system may tryto reform a new lipid bilayer in the particular cell again; however,this is both time-consuming and inefficient. In addition, a lipidbilayer may not reform in the particular cell in a subsequent trial. Asa result, the overall percentage of cells in the nanopore basedsequencing chip with properly formed lipid bilayers and nanopores (i.e.,the yield of the nanopore based sequencing chip) is reduced.

A non-destructive technique to detect a lipid bilayer formed in a cellof a nanopore based sequencing chip is disclosed. A non-destructivetechnique to detect a lipid bilayer has many advantages, includingincreasing the efficiency and yield of the nanopore based sequencingchip.

FIG. 6A illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip wherein the circuitry can be configuredto detect whether a lipid bilayer is formed in the cell without causingan already formed lipid bilayer to break down.

FIG. 6A shows a lipid membrane or lipid bilayer 612 situated between acell working electrode 614 and a counter electrode 616, such that avoltage is applied across lipid membrane/bilayer 612. A lipid bilayer isa thin membrane made of two layers of lipid molecules. A lipid membraneis a membrane made of several layers (more than two) of lipid molecules.Lipid membrane/bilayer 612 is also in contact with a bulkliquid/electrolyte 618. Note that working electrode 614, lipidmembrane/bilayer 612, and counter electrode 616 are drawn upside down ascompared to the working electrode, lipid bilayer, and counter electrodein FIG. 1. In some embodiments, the counter electrode is shared betweena plurality of cells, and is therefore also referred to as a commonelectrode. The common electrode can be configured to apply a commonpotential to the bulk liquid in contact with the lipidmembranes/bilayers in the measurements cells by connecting the commonelectrode to a voltage source V_(liq) 620. The common potential and thecommon electrode are common to all of the measurement cells. There is aworking cell electrode within each measurement cell; in contrast to thecommon electrode, working cell electrode 614 is configurable to apply adistinct potential that is independent from the working cell electrodesin other measurement cells.

FIG. 6B illustrates the same circuitry 600 in a cell of a nanopore basedsequencing chip as that shown in FIG. 6A. Comparing to FIG. 6A, insteadof showing a lipid membrane/bilayer between the working electrode andthe counter electrode, an electrical model representing the electricalproperties of the working electrode and the lipid membrane/bilayer isshown.

Electrical model 622 includes a capacitor 624 representing theelectrical properties of working electrode 614. The capacitanceassociated with working electrode 614 is also referred to as a doublelayer capacitance (C_(double layer)). Electrical model 622 furtherincludes a capacitor 626 (C_(bilayer)) that models a capacitanceassociated with the lipid membrane/bilayer and a resistor 628(R_(bilayer)) that models a resistance associated with the lipidmembrane/bilayer. The resistance associated with the lipidmembrane/bilayer is very high, and therefore R_(bilayer) may be replacedby an open circuit, which reduces electrical model 622 toC_(double layer) in series with C_(bilayer).

Voltage source V_(liq) 620 is an alternating current (AC) voltagesource. Counter electrode 616 is immersed in the bulk liquid 618, and anAC non-Faradaic mode is utilized to modulate a square wave voltageV_(liq) and apply it to the bulk liquid in contact with the lipidmembranes/bilayers in the measurement cells. In some embodiments,V_(liq) is a square wave with a magnitude of ±200-250 mV and a frequencybetween 25 and 100 Hz.

Pass device 606 is a switch that can be used to connect or disconnectthe lipid membrane/bilayer and the electrodes from the measurementcircuitry 600. The switch enables or disables a voltage stimulus thatcan be applied across the lipid membrane/bilayer in the cell. Beforelipids are deposited to the cell to form a lipid bilayer, the impedancebetween the two electrodes is very low because the well of the cell isnot sealed, and therefore switch 606 is kept open to avoid ashort-circuit condition. Switch 606 may be closed once lipid solvent hasbeen deposited to the cell that seals the well of the cell.

Circuitry 600 further includes an on-chip fabricated integratingcapacitor 608 (n_(cap)). Integrating capacitor 608 is pre-charged byusing a reset signal 603 to close switch 601, such that integratingcapacitor 608 is connected to a voltage source V_(pre) 605. In someembodiments, voltage source V_(pre) 605 provides a constant positivevoltage with a magnitude of 900 mV. When switch 601 is closed,integrating capacitor 608 is pre-charged to the positive voltage levelof voltage source V_(pre) 605.

After integrating capacitor 608 is pre-charged, reset signal 603 is usedto open switch 601 such that integrating capacitor 608 is disconnectedfrom voltage source V_(pre) 605. At this point, depending on the levelof V_(liq), the potential of counter electrode 616 may be at a higherlevel than the potential of working electrode 614, or vice versa. Forexample, during the positive phase of square wave V_(liq) (i.e., thedark period of the AC voltage source signal cycle), the potential ofcounter electrode 616 is at a higher level than the potential of workingelectrode 614. Similarly, during the negative phase of square waveV_(liq) (i.e., the bright period of the AC voltage source signal cycle),the potential of counter electrode 616 is at a lower level than thepotential of working electrode 614. Due to this potential difference,integrating capacitor 608 may be charged during the dark period of theAC voltage source signal cycle and discharged during the bright periodof the AC voltage source signal cycle.

Depending on the sampling rate of an analog-to-digital converter (ADC)610, integrating capacitor 608 charges or discharges for a fixed periodof time, and then the voltage stored in integrating capacitor 608 may beread out by ADC 610. After the sampling by ADC 610, integratingcapacitor 608 is pre-charged again by using reset signal 603 to closeswitch 601, such that integrating capacitor 608 is connected to voltagesource V_(pre) 605 again. In some embodiments, the sampling rate of ADC610 is between 1500 to 2000 Hz. In some embodiments, the sampling rateof ADC 610 is up to 5 kHz. For example, with a sampling rate of 1 kHz,integrating capacitor 608 charges or discharges for a period of ˜1 ms,and then the voltage stored in integrating capacitor 608 is read out byADC 610. After the sampling by ADC 610, integrating capacitor 608 ispre-charged again by using reset signal 603 to close switch 601 suchthat integrating capacitor 608 is connected to voltage source V_(pre)605 again. The steps of pre-charging the integrating capacitor 608,waiting a fixed period of time for the integrating capacitor 608 tocharge or discharge, and sampling the voltage stored in integratingcapacitor by ADC 610 are then repeated in cycles throughout a lipidbilayer measurement phase of the system.

Circuitry 600 may be used to detect whether a lipid bilayer is formed inthe cell by monitoring a delta voltage change, ΔV_(ADC), at integratingcapacitor 608 (n_(cap)) in response to a delta voltage change (ΔV_(liq))applied to the bulk liquid in contact with the lipid membrane/bilayer.As will be described in greater detail below, during the lipid bilayermeasurement phase, circuitry 600 may be modeled as a voltage dividerwith C_(bilayer) 626, C_(double layer) 624, and n_(cap) 608 connected inseries, and a voltage change tapped at an intermediate point of thevoltage divider can be read by ADC 610 for determining whether a lipidbilayer has been formed.

FIG. 7 illustrates an electrical model 700 representing the electricalproperties of a portion of circuitry 600 during the lipid bilayermeasurement phase of the system. As shown in FIG. 7, C_(double layer)624 is connected in series with C_(bilayer) 626, but R_(bilayer) 628(see FIG. 6B) is eliminated from electrical model 700. R_(bilayer) 628can be removed from electrical model 700 because the resistanceassociated with the lipid membrane/bilayer is very high, and thereforeR_(bilayer) may be approximated as an open circuit. As shown in FIG. 7,C_(double layer) 624 and C_(bilayer) 626 are further connected in serieswith n_(cap) 608.

When operating in an AC mode, the voltage read by the ADC (V_(ADC)) canbe determined by:

$\begin{matrix}{V_{ADC} = {V_{liq}*\frac{z({ncap})}{{z({bilayer})} + {z\left( {{double}\mspace{14mu} {layer}} \right)} + {z({ncap})}}}} & {{Equation}\mspace{14mu} (1)}\end{matrix}$

where Z=1/(jωC),

Z(ncap) is the AC impedance associated with n_(cap),

Z(double layer) is the AC impedance associated with the workingelectrode,

and Z(bilayer) is the AC impedance associated with the lipidmembrane/bilayer.

The AC impedance of the double layer, Z(double layer), has a very lowvalue compared to Z(bilayer) and Z(ncap) because C_(double layer) ismuch larger than C_(bilayer) or the capacitance of n_(cap). Therefore,substituting Z(ncap)=1/(jωC_(ncap)), Z (bilayer)=1/jωC_(bilayer), andZ(double layer)=0, equation (1) can be simplified as:

$\begin{matrix}{V_{ADC} = {V_{liq}*\frac{C({bilayer})}{{C({ncap})} + {C({bilayer})}}}} & {{Equation}\mspace{14mu} (2)}\end{matrix}$

where C(ncap) is the capacitance associated with n_(cap),

and C(bilayer) is the capacitance associated with the lipidmembrane/bilayer.

When lipids are first deposited into the cells to form the lipidbilayers, some of the cells have lipid bilayers spontaneously formed,but some of the cells merely have a thick lipid membrane (with multiplelayers of lipid molecules and solvent combined together) spanning acrosseach of the wells of the cells. The capacitance associated with a lipidbilayer is larger than the capacitance associated with a lipid membranethat is more than two layers of lipid molecules thick because thecapacitance of the lipid membrane/bilayer is inversely proportional toits thickness. As a lipid membrane thins out and transitions to become alipid bilayer, the thickness decreases and its associated capacitanceincreases. In Equation (2) above, as a lipid bilayer begins to formwithin a cell, C(bilayer) increases while C(ncap) remains constant, suchthat on the whole V_(ADC) increases. An increase in V_(ADC) cantherefore be used as an indicator that a lipid bilayer has been formedwithin a cell.

In some embodiments, a delta voltage change ΔV_(ADC) at integratingcapacitor 608 (n_(cap)) in response to a delta voltage change (ΔV_(liq))applied to the bulk liquid in contact with the lipid membrane/bilayer ismonitored in order to detect whether a lipid bilayer has been formed ina cell. For example, Equation (2) may be rewritten as:

$\begin{matrix}{{\Delta \; V_{ADC}} = {\Delta \; V_{liq}*\frac{C({bilayer})}{{C({ncap})} + {C({bilayer})}}}} & {{Equation}\mspace{14mu} (3)}\end{matrix}$

-   -   where ΔV_(ADC) is a voltage change at integrating capacitor 608        (n_(cap)) read by the ADC,    -   ΔV_(liq) is a voltage change applied to the bulk liquid,    -   C(ncap) is the capacitance associated with n_(cap),    -   and C(bilayer) is the capacitance associated with the lipid        membrane/bilayer.

In Equation (3) above, because C(ncap) remains constant, whileC(bilayer) increases as a lipid bilayer begins to form within a cell,ΔV_(ADC) increases as well. ΔV_(ADC) is roughly proportional to thecapacitance associated with the lipid membrane/bilayer, C(bilayer). Anincrease in ΔV_(ADC) can therefore be used as an indicator that a lipidbilayer has been formed within a cell.

In some embodiments, in order to maximize the observable ΔV_(ADC) for amore reliable detection of a lipid bilayer, ΔV_(ADC) in response to amaximum voltage change applied to the bulk liquid in contact with thelipid membrane/bilayer (max ΔV_(liq)) is monitored in order to detectwhether a lipid bilayer has been formed in a cell.

FIG. 8A illustrates that a small observed positive/negative voltagechange ±ΔV_(ADC) in response to a positive/negative voltage change±ΔV_(liq) results in no lipid bilayer being detected to have been formedin the cell. FIG. 8B illustrates that a large observed positive/negativevoltage change ±ΔV_(ADC) in response to a positive/negative voltagechange ±ΔV_(liq) results in the detection of a lipid bilayer having beenformed in a cell.

In FIG. 8A, a maximum positive voltage change +ΔV_(liq) occurs when thesquare wave V_(liq) changes from a negative phase to a positive phase,while a maximum negative voltage change −ΔV_(liq) occurs when the squarewave V_(liq) changes from a positive phase to a negative phase. In FIG.8A, at the instance when ΔV_(liq) is at a positive maximum, only a small+ΔV_(ADC) can be observed if a lipid bilayer has not been formed in thecell; at the instance when ΔV_(liq) is at a negative maximum, only asmall −ΔV_(ADC) can be observed if a lipid bilayer has not been formedin the cell.

In FIG. 8B, at the instance when ΔV_(liq) is at a positive maximum, alarge positive voltage change +ΔV_(ADC) can be observed if a lipidbilayer has already been formed in the cell. And at the instance whenΔV_(liq) is at a negative maximum, a large negative voltage change−ΔV_(ADC) can be observed if a lipid bilayer has already been formed inthe cell.

In some embodiments, the absolute value of ΔV_(ADC) (|ΔV_(ADC)|)observed when the absolute value of ΔV_(liq) (|ΔV_(liq)|) is at amaximum is compared with a predetermined threshold. If(|ΔV_(ADC)|>predetermined threshold), then it is determined that a lipidbilayer is detected. Conversely, if (|ΔV_(ADC)|<predeterminedthreshold), then it is determined that a lipid bilayer is not detected.

FIG. 9A illustrates an exemplary plot of V_(ADC) versus time before andafter a lipid bilayer is formed within a cell. The plot in FIG. 9A isbased on real testing data. As shown in FIG. 9A, the units of V_(ADC) onthe y-axis are in ADC counts. However, other units may be used as well.As shown in FIG. 9A, during a time period t₁ when a lipid bilayer hasnot been formed, the recorded |ΔV_(ADC)| values are smaller than thoserecorded during a time period t₂ after a lipid bilayer has been formedin the cell.

FIG. 9B illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t₁ when a lipid bilayerhas not been formed. The results shown in FIG. 9B are consistent withFIG. 8A. In FIG. 9B, a maximum +ΔV_(liq) occurs when the square waveV_(liq) changes from a negative phase to a positive phase, while amaximum −ΔV_(liq) occurs when the square wave V_(liq) changes from apositive phase to a negative phase. In FIG. 9B, at the instance whenΔV_(liq) is at a positive maximum, only a small +ΔV_(ADC) can beobserved because a lipid bilayer has not been formed in the cell; at theinstance when ΔV_(liq) is at a negative maximum, only a small −ΔV_(ADC)can be observed because a lipid bilayer has not been formed in the cell.

FIG. 9C illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t₂ when a lipid bilayerhas been formed. The results shown in FIG. 9C are consistent with FIG.8B. In FIG. 9C, at the instance when ΔV_(liq) is at a positive maximum,a large +ΔV_(ADC) can be observed between two consecutive sample pointsbecause a lipid bilayer has already been formed in the cell. At theinstance when ΔV_(liq) is at a negative maximum, a large −ΔV_(ADC) canbe observed because a lipid bilayer has already been formed in the cell.Note that shortly after the square wave V_(liq) changes from one phaseto another, ΔV_(liq) stays at zero, and V_(ADC) reduces to zero inresponse. As shown in FIG. 9C, when a lipid bilayer has already beenformed in the cell, a positive or negative spike in V_(ADC) can beobserved. The positive or negative spikes are followed by much smallerV_(ADC) values.

As described above, when the lipid solvent mixture is first depositedinto the cells to form the lipid bilayers, some of the cells have lipidbilayers spontaneously formed, but some of the cells merely have a thicklipid membrane with multiple layers of lipid molecules combined with thesolvent spanning across each of the wells of the cells. In order toincrease the yield of the nanopore based sequencing chip (i.e., thepercentage of cells in the nanopore based sequencing chip with properlyformed lipid bilayers and nanopores), the nanopore based sequencing chipmay perform additional steps to facilitate the formation of lipidbilayers in additional cells. For example, applying an electricallipid-thinning stimulus to the cells that have not had lipid bilayersformed therein yet can improve the efficiency of liquid flow above thethick lipid membranes, thereby facilitating the removal of any excesslipid solvent such that the thick lipid membranes can be thinned out andtransitioned into lipid bilayers more efficiently. Applying theelectrical lipid-thinning stimulus to the cells that have not had lipidbilayers formed therein yet will also create electrostatic forces thattend to squeeze out the excess lipid solvent and thin out the thicklipid membranes into lipid bilayers. On the other hand, the cells thathave already had lipid bilayers properly formed therein should not befurther exposed to the same electrical lipid-thinning stimulus, as theelectrical stimulus may cause some of the thin lipid bilayers to breakdown. Therefore, it is advantageous to use the non-destructive techniquedescribed in the present application to detect and separate the portionof the cells in the nanopore based sequencing chip that have lipidbilayers formed therein from the portion of the cells that do not havelipid bilayer properly formed therein yet. By dividing the cells intodifferent groups, the cells in different groups can be processeddifferently, thereby achieving greater efficiency and increasing theoverall yield of the nanopore based sequencing chip.

FIG. 10 illustrates an embodiment of a process 1000 for an improvedtechnique of forming lipid layers in the cells of a nanopore basedsequencing chip. In some embodiments, the nanopore based sequencing chipof FIG. 10 includes a plurality of cells 100 of FIG. 1. In someembodiments, the nanopore based sequencing chip of FIG. 10 includes aplurality of cells 500 of FIG. 5. In some embodiments, the nanoporebased sequencing chip of FIG. 10 includes circuitries 600 of FIGS. 6Aand 6B.

Process 1000 includes steps in which different types of fluids (e.g.,liquids or gases) are flowed through the cells of the nanopore basedsequencing chip via a flow chamber. Multiple fluids with significantlydifferent properties (e.g., compressibility, hydrophobicity, andviscosity) are flowed over an array of sensors on the surface of thenanopore based sequencing chip. For improved efficiency, each of thesensors in the array should be exposed to the fluids in a consistentmanner. For example, each of the different types of fluids should beflowed over the nanopore based sequencing chip such that the fluid maybe delivered to the chip, evenly coating and contacting each of thecells' surfaces, and then delivered out of the chip. As described above,a nanopore based sequencing chip incorporates a large number of sensorcells configured as an array. As the nanopore based sequencing chip isscaled to include more and more cells, achieving an even flow of thedifferent types of fluids across the cells of the chip becomes morechallenging.

In some embodiments, the nanopore based sequencing system that performsprocess 1000 of FIG. 10 includes an improved flow chamber having aserpentine fluid flow channel that directs the fluids to traverse overdifferent sensors of the chip along the length of the channel. FIG. 11illustrates the top view of a nanopore based sequencing system 1100 withan improved flow chamber enclosing a silicon chip that allows liquidsand gases to pass over and contact sensors on the chip surface. The flowchamber includes a serpentine or winding flow channel 1108 that directsthe fluids to flow directly above a single column (or a single row) ofsensor banks 1106 (each bank including several thousands of sensorcells) from one end of the chip to the opposite end and then directs thefluids to repeatedly loop back and flow directly above other adjacentcolumns of sensor banks, until all of the sensor banks have beentraversed at least once. As shown in FIG. 11, system 1100 includes aninlet 1102 and an outlet 1104.

With reference to FIG. 11, a fluid is directed into system 1100 throughinlet 1102. Inlet 1102 may be a tube or a needle. For example, the tubeor needle may have a diameter of one millimeter. Instead of feeding theliquid or gas directly into a wide flow chamber with a single continuousspace, inlet 1102 feeds the liquid or gas into a serpentine flow channel1108 that directs the liquid or gas to flow directly above a singlecolumn of sensor banks 1106. The serpentine channel 1108 may be formedby stacking together a top plate and a gasket with dividers 1110 thatdivide the chamber into the serpentine channel to form a flow cell, andthen mounting the flow cell on top of the chip. Once the liquid or gasflows through the serpentine channel 1108, the liquid or gas is directedup through outlet 1104 and out of system 1100.

System 1100 allows the fluids to flow more evenly on top of all thesensors on the chip surface. The channel width is configured to benarrow enough such that capillary action has an effect. Moreparticularly, the surface tension (which is caused by cohesion withinthe fluid) and adhesive forces between the fluid and the enclosingsurfaces act to hold the fluid together, thereby preventing the fluid orthe air bubbles from breaking up and creating dead zones. For example,the channel may have a width of 1 millimeter or less. The narrow channelenables controlled flow of the fluids and minimizes the amount ofremnants from a previous flow of fluids or gases.

With reference to FIG. 10, at 1002, a salt/electrolyte buffer solutionis flowed through the cells of the nanopore based sequencing chip viathe flow chamber to substantially fill the wells in the cells with thesalt buffer solution. The salt buffer solution may include one of thefollowing: lithium chloride (LiCl), sodelectium chloride (NaCl),potassium chloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂). In some embodiments, theconcentration of the salt buffer solution is 300 mM (millimolar).

At 1004, a lipid and solvent mixture is flowed through the cells of thenanopore based sequencing chip via the flow chamber. In someembodiments, the lipid and solvent mixture includes lipid molecules suchas diphytanoylphosphatidylcholine (DPhPC). In some embodiments, thelipid and solvent mixture includes decane or tridecane. When the lipidand solvent mixture is first deposited into the cells to form the lipidbilayers, some of the cells have lipid bilayers spontaneously formed,but some of the cells merely have a thick lipid membrane (with multiplelayers of lipid molecules and solvent combined together) spanning acrosseach of the wells of the cells. In order to increase the yield of thenanopore based sequencing chip (i.e., the percentage of cells in thenanopore based sequencing chip with properly formed lipid bilayers andnanopores), the nanopore based sequencing chip will repeatedly gothrough two phases, an electrical lipid-thinning stimulus phase and abuffer flowing phase, to facilitate the formation of lipid bilayers inadditional cells.

The electrical lipid-thinning stimulus phase of process 1000 includessteps 1006, 1008, and 1010. In some embodiments, during this phase steps1006, 1008, and 1010 may be performed in the order as shown in FIG. 10.In some embodiments, steps 1006, 1008, and 1010 may be performed in adifferent order. In some embodiments, the steps may be performedsimultaneously.

At 1006, the non-destructive technique described in the presentapplication is used to detect whether a lipid bilayer is formed in acell using circuitry 600 of FIG. 6A and FIG. 6B. The detection includesmonitoring a voltage change, ΔV_(ADC), at integrating capacitor 608(n_(cap)) in response to a voltage change (ΔV_(liq)) applied to the bulkliquid in contact with the lipid membrane/bilayer. Cells that have lipidbilayers detected are separated into a different group from the cellsthat do not have lipid bilayers detected. Within each of the cells withlipid bilayers detected, pass device 606 is opened in order todisconnect the lipid bilayer and the electrodes from the measurementcircuitry 600, such that the electrical lipid-thinning stimulus isdisabled from being applied to the cell.

At 1008, an electrical lipid-thinning stimulus is applied to the cellsof the nanopore based sequencing chip. Applying the electricallipid-thinning stimulus to the cells that have not had lipid bilayersformed therein yet can improve the efficiency of liquid flow above thethick lipid membranes, thereby facilitating the removal of any excesslipid solvent such that the thick lipid membranes can be thinned out andtransitioned into lipid bilayers more efficiently. Applying theelectrical lipid-thinning stimulus to the cells that have not had lipidbilayers formed therein yet will also create electrostatic forces thattend to squeeze out the excess lipid solvent and thin out the thicklipid membranes into lipid bilayers. In some embodiments, the samecircuitry 600 of FIG. 6A and FIG. 6B may be used to apply the electricallipid-thinning stimulus. The only difference in the setup of circuitry600 between lipid bilayer detection and lipid thinning is that theabsolute magnitude of V_(liq) is lower for lipid bilayer detection. Forexample, the absolute magnitude V_(liq) for lipid bilayer detection maybe between 100 mV to 250 mV, while the absolute magnitude V_(liq) forlipid thinning may be between 250 mV to 500 mV.

At step 1010, it is determined whether the electrical lipid-thinningstimulus phase is finished. In some embodiments, the electricallipid-thinning stimulus is applied to any cells that have not beendetected as having lipid bilayers therein for a period of two seconds.However, other predetermined period of time may be used as well. If thephase is not over yet, then process 1000 returns to steps 1006 and 1008again until the time period is finished; otherwise, process 1000proceeds to the salt buffer solution flowing phase next.

The salt buffer solution flowing phase of process 1000 includes steps1012, 1014, and 1016. In some embodiments, during this phase steps 1012,1014, and 1016 may be performed in the order as shown in FIG. 10. Insome embodiments, steps 1012, 1014, and 1016 may be performed in adifferent order. In some embodiments, the steps may be performedsimultaneously.

At 1012, the same non-destructive technique used at step 1006 is used todetect whether a lipid bilayer is formed in the cell using circuitry 600of FIG. 6A and FIG. 6B. Cells that have lipid bilayers detected areseparated into a different group from the cells that do not have lipidbilayers detected. Within each of the cells with lipid bilayersdetected, pass device 606 is opened in order to disconnect the lipidbilayer and the electrodes from the measurement circuitry 600.

At 1014, a salt/electrolyte buffer solution is flowed through the cellsof the nanopore based sequencing chip via the flow chamber. The purposeof flowing the salt buffer solution over the cells is to facilitate theformation of a lipid bilayer over each of the cells. When the saltbuffer solution is flowed over the cells, the thickness of the lipid andsolvent mixture deposited on the cell is reduced, facilitating theformation of the lipid bilayer.

At 1016, it is determined whether the salt buffer solution flowing phaseis over. In some embodiments, salt buffer solution is flowed for aperiod of two seconds. However, other predetermined period of time maybe used as well. If the phase is not over yet, then process 1000 returnsto steps 1012 and 1014 again until the time period is finished;otherwise, process 1000 proceeds to step 1018.

At 1018, it is determined whether the electrical lipid-thinning stimulusphase and salt buffer solution flowing phase of process 1000 should berepeated. Different criteria may be used at this step. In someembodiments, the electrical lipid-thinning stimulus phase and saltbuffer solution flowing phase are performed a predetermined number oftimes. In some embodiments, the two phases are repeated until a targetyield for the nanopore based sequencing chip has been reached. In someembodiments, if the incremental number or percentage of cells that havejust been detected as having lipid bilayers formed during the last roundof thinning by the stimulus and the buffer solution flow is lower than apredetermined threshold, then process 1000 is terminated. In someembodiments, the two phases are repeated until the most recently appliedelectrical lipid-thinning stimulus level has reached a predeterminedmaximum threshold, e.g. 500 mV.

Process 1000 proceeds to step 1020 if the electrical lipid-thinningstimulus phase and salt buffer solution flowing phase of process 1000are going to be repeated next. At step 1020, the next electricallipid-thinning stimulus to be applied is determined. In someembodiments, the electrical lipid-thinning stimulus level is increasedby a fixed predetermined amount, e.g., an increment of 100 mV. In someembodiments, if the incremental number or percentage of cells that havejust been detected as having lipid bilayers formed during the lastiteration is lower than a predetermined threshold, then the electricallipid-thinning stimulus level is increased by a fixed predeterminedamount; otherwise, the previous electrical lipid-thinning stimulus isfound to be effective and thus the same electrical lipid-thinningstimulus level is used again.

FIGS. 12A, 12B, and 12C are histograms that illustrate that as theelectrical lipid-thinning stimulus phase and salt buffer solutionflowing phase of process 1000 repeats a number of times, the overallpercentage of cells in the nanopore based sequencing chip with properlyformed lipid bilayers (i.e., the yield of the nanopore based sequencingchip) increases. For each of the figures, the x-axis is the voltagechange at integrating capacitor 608 (n_(cap)), ΔV_(ADC), in response toa voltage change (ΔV_(liq)) applied to the bulk liquid in contact withthe lipid membrane/bilayer, while the y-axis is the number of cells withits ΔV_(ADC) value within certain ΔV_(ADC) bins. FIG. 12A illustratesthe initial distribution of cells with different ΔV_(ADC) values. FIG.12B illustrates the distribution of cells with different ΔV_(ADC) valuesafter the lipid-thinning stimulus phase and salt buffer solution flowingphase of process 1000 have repeated a number of times. FIG. 12Cillustrates the distribution of cells with different ΔV_(ADC) valuesafter the lipid-thinning stimulus phase and salt buffer solution flowingphase of process 1000 have repeated an even greater number of times. Inthis example, cells that have a ΔV_(ADC) value of 50 or above aredetermined as having lipid bilayers formed therein. As shown in FIG.12A, initially, only a small number of cells have lipid bilayersdetected. As shown in FIG. 12B, after the lipid-thinning stimulus phaseand salt buffer solution flowing phase of process 1000 have repeated anumber of times, the number of cells having lipid bilayers detectedincreases. Finally, as shown in FIG. 12C, after the lipid-thinningstimulus phase and salt buffer solution flowing phase of process 1000have repeated an even greater number of times, a high majority of thecells in the nanopore based sequencing chip has lipid bilayers detected.

FIG. 13 illustrates an embodiment of a timing diagram for a bilayermeasurement phase, an electrical lipid-thinning stimulus phase, and asalt buffer solution flowing phase. In this example, after lipids aredeposited into the cells, the bilayer measurement phase is started. Thebilayer measurement phase lasts about 2 seconds in time. During thisphase, all of the cells are enabled. The absolute value of V_(liq) is250 mV.

The bilayer measurement phase is followed by the electricallipid-thinning stimulus phase, which lasts about 2 seconds in time.During this phase, when the absolute value of ΔV_(ADC) (|ΔV_(ADC)|)exceeds threshold1 within a cell, then a lipid bilayer is detectedwithin the cell and the cell is disconnected from the voltage source.The absolute value of V_(liq) is between 250-500 mV.

The electrical lipid-thinning stimulus phase is followed by the saltbuffer solution flowing phase, and the latter lasts about 2-10 secondsin time. During this phase, when the absolute value of ΔV_(ADC)(|ΔV_(ADC)|) exceeds threshold2 within a cell, then a lipid bilayer isdetected within the cell and the cell is disconnected from the voltagesource. The absolute value of V_(liq) is 250 mV.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

What is claimed is:
 1. A method of detecting a lipid bilayer formed in acell of a nanopore based sequencing chip, comprising: coupling a lipidmembrane with an integrating capacitor, wherein the lipid membrane isbetween a working electrode and a counter electrode; applying analternating current (AC) voltage to the counter electrode; periodicallysampling a voltage across the integrating capacitor by ananalog-to-digital converter (ADC); determining a change in the sampledvoltage across the integrating capacitor in response to a change in theAC voltage; and detecting that the lipid membrane comprises a lipidbilayer based on the determined change in the sampled voltage across theintegrating capacitor in response to the change in the AC voltage. 2.The method of claim 1, further comprising determining the change in thesampled voltage across the integrating capacitor in response to thechange in the AC voltage when the AC voltage changes from a positivephase to a negative phase or when the AC voltage changes from a negativephase to a positive phase.
 3. The method of claim 2, further comprisingdetermining the change in the sampled voltage across the integratingcapacitor in response to the change in the AC voltage when the ACvoltage changes from a positive phase to a negative phase or when the ACvoltage changes from a negative phase to a positive phase, and whereinan AC positive phase comprises a positive square wave and an AC negativephase comprises a negative square wave.
 4. The method of claim 1,further comprising detecting that the lipid membrane comprises a lipidbilayer by detecting a spike in the sampled voltage across theintegrating capacitor followed by a substantially smaller sampledvoltage across the integrating capacitor, and wherein the spike occurswhen the AC voltage changes from a positive phase to a negative phase orwhen the AC voltage changes from a negative phase to a positive phase.5. The method of claim 4, further comprising comparing a magnitude ofthe spike in the sampled voltage across the integrating capacitoragainst a predetermined threshold, and in the event that the magnitudeof the spike is above the predetermined threshold, detecting that thelipid membrane comprises a lipid bilayer.
 6. The method of claim 1,further comprising: pre-charging the integrating capacitor by connectingthe integrating capacitor to a constant pre-charging voltage source;after the integrating capacitor is charged to the constant pre-chargingvoltage source value, disconnecting the pre-charging voltage source fromthe integrating capacitor; waiting a predetermined period of time forthe integrating capacitor to charge or discharge through the capacitanceassociated with the lipid membrane; and sampling the voltage across theintegrating capacitor after the predetermined waiting period.
 7. Themethod of claim 6, wherein the steps of pre-charging the integratingcapacitor, disconnecting the pre-charging voltage source from theintegrating capacitor, and waiting for the integrating capacitor tocharge or discharge through the capacitance associated with the lipidmembrane are repeated at the sampling rate of the ADC.
 8. The method ofclaim 7, wherein the sampling rate of the ADC is at least ten timeshigher than a frequency of the AC voltage.
 9. The method of claim 1,further comprising: in response to the detection that the lipid membranecomprises a lipid bilayer, disabling further electrical stimuli frombeing applied across the lipid membrane by opening a switch in the cellsuch that the lipid bilayer does not break down due to an electricfield.
 10. The method of claim 1, further comprising: disablingelectrical stimuli from being applied across the working electrode andthe counter electrode before the lipid membrane has sealed a well withinthe cell by opening a switch in the cell.
 11. A system for detecting alipid bilayer formed in a cell of a nanopore based sequencing chip,comprising: an integrating capacitor; a working electrode coupled to theintegrating capacitor; a counter electrode, wherein a lipid membranedeposited between the working electrode and the counter electrode iscoupled with the integrating capacitor; an alternating current (AC)voltage source that applies an AC voltage to the counter electrode; ananalog-to-digital converter (ADC) periodically sampling a voltage acrossthe integrating capacitor; and a processor or a circuitry configured to:determine a change in the sampled voltage across the integratingcapacitor in response to a change in the AC voltage; and detect that thelipid membrane comprises a lipid bilayer based on the determined changein the sampled voltage across the integrating capacitor in response tothe change in the AC voltage.
 12. The system of claim 11, wherein theprocessor or circuitry is further configured to: determine the change inthe sampled voltage across the integrating capacitor in response to thechange in the AC voltage when the AC voltage changes from a positivephase to a negative phase or when the AC voltage changes from a negativephase to a positive phase.
 13. The system of claim 12, wherein theprocessor or circuitry is further configured to: determine the change inthe sampled voltage across the integrating capacitor in response to thechange in the AC voltage when the AC voltage changes from a positivephase to a negative phase or when the AC voltage changes from a negativephase to a positive phase, and wherein an AC positive phase comprises apositive square wave and an AC negative phase comprises a negativesquare wave.
 14. The system of claim 11, wherein the processor orcircuitry is further configured to: detect that the lipid membranecomprises a lipid bilayer by detecting a spike in the sampled voltageacross the integrating capacitor followed by a substantially smallersampled voltage across the integrating capacitor, and wherein the spikeoccurs when the AC voltage changes from a positive phase to a negativephase or when the AC voltage changes from a negative phase to a positivephase.
 15. The system of claim 14, wherein the processor or circuitry isfurther configured to: compare a magnitude of the spike in the sampledvoltage across the integrating capacitor against a predeterminedthreshold, and in the event that the magnitude of the spike is above thepredetermined threshold, detect that the lipid membrane comprises alipid bilayer.
 16. The system of claim 11, further comprising: aconstant pre-charging voltage source; wherein the processor or circuitryis further configured to: pre-charge the integrating capacitor byconnecting the integrating capacitor to the constant pre-chargingvoltage source; after the integrating capacitor is charged to theconstant pre-charging voltage source value, disconnect the pre-chargingvoltage source from the integrating capacitor; wait a predeterminedperiod of time for the integrating capacitor to charge or dischargethrough the capacitance associated with the lipid membrane; and causethe ADC to sample the voltage across the integrating capacitor after thepredetermined waiting period.
 17. The system of claim 16, wherein thesteps of pre-charging the integrating capacitor, disconnecting thepre-charging voltage source from the integrating capacitor, and waitingfor the integrating capacitor to charge or discharge through thecapacitance associated with the lipid membrane are repeated at thesampling rate of the ADC.
 18. The system of claim 17, wherein thesampling rate of the ADC is at least ten times higher than a frequencyof the AC voltage.
 19. The system of claim 17, wherein the sampling rateof the ADC is between 1500 to 5000 Hz, and wherein a frequency of the ACvoltage is between 25 and 100 Hz.
 20. The system of claim 11, furthercomprising: a switch in the cell controlled by the processor or thecircuitry; wherein the processor or circuitry is further configured to:in response to the detection that the lipid membrane comprises a lipidbilayer, disable further electrical stimulus from being applied acrossthe lipid membrane by opening the switch in the cell.
 21. The system ofclaim 11, further comprising: a switch in the cell controlled by theprocessor or the circuitry; wherein the processor or circuitry isfurther configured to: disable electrical stimuli from being appliedacross the working electrode and the counter electrode before the lipidmembrane has sealed a well within the cell by opening the switch in thecell.
 22. The system of claim 11, wherein an absolute magnitude of theAC voltage is between 100 and 250 mV.